In an era of HIV, multidrug resistant tuberculosis, and bioterrorism, the need for novel chemotherapeutic agents against Mycobactedum tuberculosis has never been greater. The durable success of M. tuberculosis as a pathogen is largely attributable to its ability to evade a sterilizing response from the innate immune system through the evolution of mechanisms that either defuse or repair noxious insults. A detailed understanding of the antimicrobial mechanisms used by the innate immune system, therefore, may reveal novel chemotherapeutic targets. Macrophage-associated nitric oxide and reactive nitrogen intermediates (NO/RNI) are a critical component of this innate immune response. Surprisingly, no specific protein targets of NO/RNI-mediated damage have been identified. The focus of the proposed work is to define target of NO/RNI-mediated protein-S-nitrosylation and novel effects of NO/RNI on biotinylated proteins of M. tuberculosis. Accumulating evidence supports an emerging role for protein-S-nitrosylation as an important mediator of the antimicrobial effects of NO/RNI. The obligatory role of biotin in lipid biosynthesis and the importance of lipids in the virulence of M. tuberculosis suggest a similarly important antimicrobial role for the NO/RNI-dependent effects discovered in our preliminary studies. We propose to evaluate the importance of these NO/RNI-dependent effects using the 'biotin-switch' method to identify, at the proteomic level, Snitrosylated proteins; biotin-based affinity chemistries to study NO-induced alterations in biotinylated proteins; and a combination of enzyme assays, chemical inhibitor studies and macrophage infection experiments using site-directed mutagenesis and targeted gene knockouts to assess the functional importance of these targets. The identification of bona fide targets of NOIRNI-mediated damage in M. tuberculosis offers the promise of developing novel antitubercular agents that augment or mimic the natural immune response to infection with M. tuberculosis while circumventing NO/RNI-specific defenses.